Influenza virus causes one of the most important respiratory viral diseases in humans, with significant medical and economic burdens. Approximately 10% to 20% of the world population is estimated to be infected during seasonal epidemics. Influenza virus causes 250,000-500,000 deaths worldwide annually; a global pandemic could kill millions (Osterholm, M. T. N Engl J Med. 2005 352:1839-1842; Viboud, C., et al. PLoS Curr 2010 RRN1153). In the US, influenza kills an average of 17,000-51,000 people in the United States (US) per year, causes an average of over 100,000 influenza-related hospitalizations and results in an economic cost of $12 billion per year (Thompson, W. W., et al. JAMA 2004 292:1333-1340).
Influenza is a lipid-enveloped virus with a segmented negative sense RNA genome, which belongs to the family Orthomyxoviridae. The envelope of the virion contains two types of surface glycoproteins, which play essential roles in viral infection. The hemagglutinin (HA) is responsible for attachment of the virus to sialic acid-containing receptors and viral entry by membrane fusion, whereas the neuraminidase (NA) is a receptor-destroying enzyme which plays important roles in viral release and cell-to-cell spread (Matrosovich, M. N., et al. J Virol 2004 78:12665-12667; Palese, P., et al. J Gen Virol 1976 33:159-163). There are 16 identified HA subtypes and 9 recognized NA subtypes. All of these subtype combinations have been isolated in birds. Currently circulating influenza viruses in human populations contain HA and NA combinations out of three different HA subtypes (H1, H2 and H3) and 2 different NA (N1 and N2) subtypes. However, there are often outbreaks of transmissions of avian host derived influenza viruses to human population from the poultry farms (Abdel-Ghafar, A. N., et al. N Engl J Med 2008 358:261-273).
Influenza viruses undergo changes over time, allowing them to evade the host immune system and to reduce the effectiveness of immunity to prior infections or vaccinations. Influenza A viruses can change by two different means: “antigenic drift” and “antigenic shift.” Point mutations in the HA and/or NA antigens generate antigenically new influenza viruses with antigenic drift that occur during viral replication. The regular recurrence of influenza epidemics is thought to be caused by antigenic drift. Over some years sufficient changes accumulate in the virus to allow repeated infections of the host with antigenically different influenza viruses. These “major antigenic shifts” result in novel antigenic subtypes of the HA and/or NA glycoproteins that had not previously infected most of the human population, and therefore can spread rapidly causing global disease pandemics. Three global pandemics of influenza occurred during the 20th century, and were caused by H1N1 subtype viruses in 1918, H2N2 viruses in 1957, and H3N2 viruses in 1968. In addition to the circulating human influenza subtypes, other avian origin influenza viruses including H5N1, H7N2, H7N3, H7N7 and H9N2 subtypes have been shown to cause human infections on multiple occasions (Cheung, C. L., et al. J Infect Dis 2006 193:1626-1629; de Jong, M. D., et al. N Engl J Med 353:2667-2672 2005; Fouchier, R. A., et al. Proc Natl Acad Sci USA 2004 101:1356-1361; Le, Q. M., et al. Nature 2005 437:1108; Peiris, M., et al. Lancet 1999 354:916-917; Wong, S. S., et al. Chest 2006 129:156-168). The emergence or re-emergence of highly pathogenic avian influenza H5N1 viruses in domestic poultry and the increasing numbers of direct transmission of avian viruses to humans underscore a persistent threat to public health (Claas, E. C., et al. Vaccine 1998 16:977-978; Subbarao, K., et al. Science 1998 279:393-396). Most recently, the 2009 outbreak of a new H1N1 virus illustrates how fast a new pandemic virus can spread in the human population once it acquires the ability to transmit among humans (Nava, G. M., et al. Euro Surveill 2009 14; Solovyov, A., et al. Euro Surveill 2009 14).
Inactivated influenza A and B virus vaccines have been extensively used in humans. The vaccines consist of purified virus that has been chemically inactivated with formalin or β-propiolactone, and in most vaccines the virus is also detergent-treated to produce soluble forms of the viral surface antigens. Influenza epidemics in human population contain two influenza A subtypes (H1N1 and H3N2) and one variant of influenza B virus, which become major components of being a trivalent current influenza vaccine. As an alternative approach to influenza immunization, live attenuated influenza virus (LAIV) vaccines administered by nasal spray (FluMist®) have been successfully developed. The vaccine is trivalent, containing influenza virus reassortants of the strains recommended for the current season. The currently used influenza vaccines induce immune responses to the viral surface glycoproteins HA and NA; although protective, the immunity is highly strain specific. Because these proteins undergo extensive antigenic variation, frequent changes are necessary in the vaccine composition. Although the current vaccines include proteins of the two currently circulating subtypes of influenza A viruses, they are not effective in protecting against the spectrum of different antigenic subtypes of influenza A viruses that are abundant in avian species which could potentially cause new influenza pandemics in humans.
Drifted strains that are not matched with the seasonal vaccine can appear following annual formulation of the vaccine composition, significantly compromising the vaccination efficacy. It has been suggested that approximately once every decade the mismatch between virus and vaccine is high enough to reduce vaccine effectiveness by 70%. The major limitations of the current vaccines include the need to produce new vaccines every season, the uncertainty in choice of the correct strains, long production times as well as the fact that the vaccines are produced by a slow process requiring embryonated eggs. Improved vaccines are needed, not only for seasonal influenza, but also for a potential influenza pandemic.
In contrast to HA, the influenza A M2 protein has a highly conserved extracellular domain of 23 amino acids (M2e). However, due to its small size and low immunogenicity, previous studies have focused on M2e peptide fusion constructs using a variety of carrier molecules: hepatitis B virus core (De Filette, M., et al. Vaccine 2006 24:544-551; Fan, J., et al. Vaccine 2004 22:2993-3003; Neirynck, S., et al. Nat Med 1999 5:1157-1163), human papillomavirus L protein (Ionescu, R. M., et al. J Pharm Sci 2006 95:70-79), keyhole limpet hemocyanin (Tompkins, S. M., et al. Emerg Infect Dis 2007 13:426-435), bacterial outer membrane complex (Fan, J., et al. Vaccine 2004 22:2993-3003; Fu, T. M., et al. Vaccine 2009 27:1440-1447), liposome (Ernst, W. A., et al. Vaccine 2006 24:5158-5168), and flagellin (Huleatt, J. W., et al. Vaccine 2008 26:201-214). M2 vaccines based on M2e fusion carriers or combinations of M2 expressing DNA and recombinant vectors were reported to provide cross protection against lethal infection with different strains (Ernst, W. A., et al. Vaccine 2006 24:5158-5168; Fan, J., et al. Vaccine 2004 22:2993-3003; Frace, A. M., et al. Vaccine 1999 17:2237-2244; Tompkins, S. M., et al. Emerg Infect Dis 2007 13:426-435). These studies suggested that M2e antibodies played an important role in providing protection. However, previous studies on M2e conjugate vaccines used potent adjuvants such as cholera toxins or heat labile endotoxins' derivatives, saponin QS21, Freund's adjuvants, or bacterial protein conjugates (De Filette, M., et al. Vaccine 2006 24:544-551; Eliasson, D. G., et al. Vaccine 2008 26:1243-1252; Fan, J., et al. Vaccine 2004 22:2993-3003; Fu, T. M., et al. Vaccine 2009 27:1440-1447; Huleatt, J. W., et al. Vaccine 2008 26:201-214; Mozdzanowska, K., et al. Virol J 2007 4:118). Such adjuvants that nonspecifically elicit host responses including inflammation and undesirable side effects are potentially adverse in developing a widely applicable prophylactic influenza vaccine. Moreover, the longevity and breadth of cross-protection mediated by M2 immunity remain unknown.